1. Field of the Invention
The present invention relates generally to methods for nucleic acid detection. More specifically, embodiments of the invention relate to a polynucleotide that acts as a “marker” for the presence of the same or a different polynucleotide of interest. In such embodiments, methods rely on the use of organophosphate hydrolase activity as a marker which has both selectable and screenable properties.
2. Description of the Related Art
Methods to detect nucleic acids and to detect specific nucleic acids provide a foundation upon which the large and rapidly growing field of molecular biology is built. There is constant need for alternative methods and products. The reasons for selecting one method over another are varied, and include a desire to avoid radioactive materials, the lack of a license to use a technique, the cost or availability of reagents or equipment, the desire to minimize the time spent or the number of steps, the accuracy or sensitivity for a certain application, the ease of analysis, or the ability to automate the process.
The detection of nucleic acids or specific nucleic acids is often a portion of a process rather than an end in itself. There are many applications of the detection of nucleic acids in the art, and new applications are always being developed. The ability to detect and quantify nucleic acids is useful in detecting microorganisms, viruses and biological molecules, and thus affects many fields, including human and veterinary medicine, food processing and environmental testing. Additionally, the detection and/or quantification of specific biomolecules from biological samples (e.g., tissue, sputum, urine, blood, semen, saliva) has applications in forensic science, such as the identification and exclusion of criminal suspects and paternity testing as well as in genetics and medical diagnostics.
However, many attempts have been made to genetically engineer desired traits into genomes by introduction of exogenous genes using genetic engineering techniques. An important aspect of the success achieved in genetic engineering has been the ability to select or screen for transgenic cells. Most of the first successes in genetic engineering relied on utilization of selectable markers for identification of transgenic cells. Markers which have been used for selection of transgenic cells include, for example, genes that confer resistance to antibiotics and other-toxins, including, for example, ampicillin, neomycin, puromycin, methotrexate or tetracycline, those that complement auxotrophic deficiencies, or those supply critical nutrients not available from complex media. Other selectable markers include the dihydrofolate reductase gene, which confers resistance to methotrexate, and thymidine kinase, or genes conferring resistance to G418 or hygromycin. Commonly used selectable markers for plant transformation include a neomycin phosphotransferase gene (Potrykus et al., Mol. Gen. Genet. 199:183 (1985)), which provides resistance to kanamycin, paromomycin and G418; a bar gene which codes for bialaphos or phosphinothricine resistance (U.S. Pat. No. 5,550,318); a mutant aroA gene which encodes an altered EPSP synthase protein conferring glyphosate resistance (Hinchee et al., Bio/Technology 6:915–22 (1988)); a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., J. Biol. Chem. 263:6310–14 (1988)); a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone, sulfonylurea or other ALS inhibiting chemicals (European Patent Application No. 154,204, 1985); a methotrexate resistant DHFR gene (Thillet et al., J. Biol. Chem. 263:12500–08 (1988)); a dalapon dehalogenase gene that confers resistance to the herbicide dalapon; and a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan.
More recently, interest has increased in utilization of screenable or scorable markers. A screenable or scorable marker is a gene that codes for a protein whose activity is easily detected, allowing cells expressing such a marker to be readily identified. Such screenable markers include a β-glucuronidase, or uidA gene (GUS), which encodes an enzyme for which various chromogenic substrates are known; chloramphenicol acetyl transferase; alkaline phosphatase; a R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., in CHROMOSOME STRUCTURE AND FUNCTION, Kluwer Academic Publishers, Appels and Gustafson eds., pp. 263–282 (1988)); a p-lactamase gene (Sutcliffe, Proc. Nat'l. Acad. Sci. U.S.A. 75:3737 (1978)), which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky et al., Proc. Nat'l. Acad. Sci. U.S.A. 80:1101 (1983)), which encodes a catechol dioxygenase that can convert chromogenic catechols; an a-amylase gene (Ikuta et al., Biotech. 8:241 (1990)); a tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703 (1983)), which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form the easily detectable compound melanin; a β-galactosidase gene, which encodes an enzyme for which there are chromogenic substrates; a lux gene, which encodes a luciferase, the presence of which may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry; and a green fluorescent protein (GFP) gene (Sheen et al., Plant J. 8(5):777–84 (1995)).
Despite the abundance of selectable and screenable markers for genetic engineering, there are very few, if any, markers which have both selectable and screenable properties. It would be beneficial if another marker were available for detecting the presence of a polynucleotide. It is therefore an object of the present invention to provide methods for determining whether a cell has incorporated a polynucleotide using a marker which has selectable and/or screenable properties.